Solid nanoparticle with inorganic coating

ABSTRACT

A nanoparticle having a solid core comprising a biologically active substance, said core being enclosed by an inorganic coating, a method for preparing the nanoparticle, and the use of the nanoparticle in therapy. A kit comprising the nanoparticle and a pharmaceutical composition comprising the nanoparticle.

FIELD OF THE INVENTION

The present invention relates to the field of nanoparticle technology.In particular, the invention relates to solid nanoparticles for use inthe pharmaceutical field, e.g. for drug delivery.

BACKGROUND OF THE INVENTION

Many of today's drugs are formulated in the solid state and an oftenencountered problem is the poor water solubility of such drugs, whichnot only renders the drug difficult to formulate, but also may pose anobstacle to an adequate biodistribution in the body of the patient.Various methods have been developed for enhancing the bioavailability ofsuch poorly soluble drugs. One method is to formulate the drug innanoparticulate form.

With a decrease in particle size, and a consequent increase in ratio ofsurface area/mass, the rate of dissolution is enhanced. While the smallsize scale of the particles is considered to enhance dissolution rate,there still may be a problem of allowing the particle to reach itsdesired target in the body before dissolution takes place. Furthermore,while generally it is considered that the small size of the particleswill allow the particles to penetrate barriers such as cell membraneswithin the human and animal body, targeted delivery nonethelessgenerally requires the particles to be provided with adequate surfacefunctionalizations and terminations, in addition to protection againstpremature dissolution or disintegration in the body.

Generally, particles having a size of from 0.1 μm (micrometer), i.e. 100nm (nanometers) to 100 μm, i.e. 100 000 nm, are classified asmicroparticles, whereas particles having a size of from 1 to 100 nm aregenerally defined as nanoparticles. For the purpose of the presentinvention, the term “nanoparticle” will be used to designate both typesof particles, unless otherwise specifically indicated or apparent fromthe context.

There is an ever increasing demand of advanced and controlled drugdelivery, i.e., use of formulation components and devices to release atherapeutic agent at a predictable rate in vivo when administered by aninjected or non-injected route. Some drugs have an optimum concentrationrange and the controlled delivery should be designed for that range toachieve effective therapies and also reducing/eliminating potential forboth under- and overdosing. Besides keeping the drug concentration inthe body constant for a long time, there might be needs of cycling thedelivery over a long period of time or trigger drug release. Finally,effectiveness of a drug and cellular uptake can be improved considerablyby functionalizing and attachment of target molecules to the drugmolecules.

The direct delivery of drugs and biomolecules is generally inefficientand can seldom meet the requirements mentioned above. Hence, moreeffective drug transport and release systems, including different kindsof vehicles, have been designed and used. Polymers, liposomes,dendrimers and micelles are all examples of such vehicles.

A significant proportion of drugs on the market are poorly soluble inwater, and it is expected that this will be even more pronounced in thefuture. Formulations of poorly water-soluble compounds offer a challengeto the formulation experts, from the early discovery phase through thedevelopment to the launch of the pharmaceutical product.

A frequently overlooked alternative to conventional vehicles arenanoparticles. However, there are some problems with the use ofnanoparticles as a drug vehicle, such as particle aggregation andOstwald ripening (growth of bigger particles at the expense of thesmaller ones).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a versatilecontrolled drug delivery system meeting the requirements mentionedabove. The drug delivery system of the present invention is based on aninorganic capsule, preferably an oxide, that totally encapsulates thedrug. The thickness of the layer can be varied and controlled down to amonolayer of atoms, meaning that the drug release can be controlled withthe layer thickness. By making a blend of drug vehicles with differentlayer thicknesses a desired drug release profile can be designed.

Some advantages of the drug delivery system of the present invention arethe possibility of providing:

-   -   controlled drug release over longer times and even cyclic,        making efficient use of drugs as well as reducing side effects;    -   capacity of of tailoring surface properties and size of the drug        vehicle for targeting of different substances, enabling the        vehicles to pass barriers like the blood-brain, placenta and        gastrointestinal barriers;    -   poorly water-soluble drugs can be transformed to water-soluble        by applying a thin and completely intact layer of an appropriate        oxide;    -   an extremely high drug load can be achieved, usually higher than        80%;    -   solid drugs can advantageously be used;    -   the encapsulating material is biodegradable;    -   the formulation routes of drugs can be standardized since        different kinds of drug particles can be provided with the same        layer or shell, facilitating the handling of drug and reducing        formulation cost;    -   the shelf life can be prolonged due to the encapsulation;    -   the whole drug delivery system is composed of constituents that        are easily excreted from the body of the treated subject after a        relative short period of time.    -   the drug release time can be varied over a wide range (from        minutes to week).

During the process of developing a nanoparticle based drug deliverysystem, the present inventors prepared nanoparticles comprising a solidcore and an inorganic coating and subjected them to various tests.

In contact with a solvent the nanoparticles however generally were foundto dissolve too rapidly, thereby being unsuitable for controlleddelivery of the active ingredient present in the solid core.

Several hypotheses for this unsatisfactory behaviour were put forwardand numerous tests were made. As a result, the inventors found that theunsatisfactory dissolution profile was due to an incomplete coverage ofthe particle surface. Indeed, the inventors found that after applyingthe inorganic coating to the nanoparticles using conventional ALD, eachsolid core was only partially coated and the coating was interrupted byholes, even when submitting the nanoparticles to several ALD coatingsteps.

The inventors surmised that the disruptions (holes) in the coating mightcorrespond to points of contact between the particles during theapplication of the coating. To verify this hypothesis, the nanoparticleswere again submitted to several ALD coating steps, and in between eachcoating, the particles were submitted to sonication. The sonicationtreatment resulted in an agitation of the particles, which in turn ledto a disaggregation of the nanoparticles. In the subsequent coatingstep, the probability was high that each particle would present at leastpartly a different surface to the ALD coating treatment, and at leastpart of the contact holes at the surface of any one particle would thusbecome covered in the subsequent treatment. By repeating the steps ofagitation and surface coating, a plurality of particles finally wasobtained wherein at least some of the particles were covered by anintegral surface coating.

The nanoparticles obtained by such a repeated treatment were found toshow an excellent profile of delayed release of the active substancepresent in the solid core.

Thus, it was realized that in order to have a non-interrupted coatingenclosing completely the solid core, application of inorganic materialmust be performed by a method including an agitation treatment of theparticles, either intermittently or continuously.

A first aspect therefore is a method of preparing a coated nanoparticlehaving a core enclosed by an in inorganic coating, the core comprising abiologically active substance; the method comprising

applying one or more layers of inorganic material to a plurality of saidsolid cores by an application method wherein the inorganic material, orprecursors for forming the inorganic material, is/are present in gasphase, andsubmitting said solid cores to agitation during and/or in betweenapplication of the layer(s) of inorganic material.

Thus, the method of applying the inorganic material coating to the solidcore is a so-called gas phase technique.

In the method of the invention, the solid cores are not present in aliquid phase during the application of inorganic material, i.e. themethod may be referred to as a gas phase method. In other words, in thecoating method of the present invention, the inorganic material orprecursors for forming the inorganic material is/are present in gasphase.

In some embodiments, more than one layer of inorganic material isapplied to the solid cores and the solid cores are submitted toagitation after applying at least one layer of inorganic material andbefore applying at least one subsequent layer of inorganic material.

In one embodiment, the method of the invention comprises

(i) applying inorganic material to a plurality of solid cores comprisinga biologically active substance,(ii) submitting the plurality of solid cores to agitation,(iii) repeating step (i) at least once.

In one embodiment, the method of the invention comprises

(i) applying inorganic material to a plurality of the solid cores,(ii) submitting the plurality of said solid cores to agitation,(iii) repeating step (i) n times, wherein n is an integer of at least 1,and(iv) when n is an integer of at least 2, repeating step (ii) after atleast some of the steps (i).

For example, n may be 2, 3, 4, 5, 6, 7, 8 or 9, and may even be as highas, e.g., 10, 20, or 30, 40 or 50 or higher.

In a second aspect there is provided a plurality of nanoparticles, eachhaving a solid core comprising a biologically active substance, saidcore being enclosed by an inorganic coating.

The plurality of nanoparticles of the invention may be used in variousapplications. For example, according to one aspect, when thebiologically active substance is a drug, the nanoparticles of thepresent inventions are provided for use in therapy. In view of this, theinvention also relates to a pharmaceutical composition comprising aplurality of therapeutically useful nanoparticles according to theinvention and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method of preparing apharmaceutical composition, said method comprising combining a pluralityof therapeutically useful nanoparticles according to the invention and apharmaceutically acceptable carrier.

In some embodiments, the nanoparticle comprises one or more intermediarylayers of a chemical composition different from that of the solid core,at the interface between the solid core and the inorganic coating.

In some further embodiments, the nanoparticle comprises one or morechemical moieties attached to the outer surface of the inorganiccoating.

In some embodiments, the step of applying inorganic material to aplurality of the solid cores, comprises

(a) introducing a first precursor, which is in a gaseous state, into areactor pre-filled with the solid nanoparticles (the solid cores) to becoated;(b) purging and/or evacuating the reactor to remove the non-reacted ornon-adsorbed precursor and the gaseous reaction by-products;(c) exposing the nanoparticles to a second precursor to activate thesurface again for the reaction of the first precursor;(d) purging and/or evacuating of the reactor and optionally repeatingthe steps (a) to (d) in order to achieve the desired coating thickness.

This is a general Atomic Layer Deposition (ALD) process, well-known tothe person of ordinary skill in the art. The steps a-d represent areaction cycle or just cycle.

In some embodiments, the method comprises processing a biologicallyactive substance, e.g. by micronizing, so as to provide a nanoparticleas a solid core to be coated, said core containing or consisting of thebiologically active substance.

In some embodiments, the method comprises applying a surface treatmentto the solid core prior to applying the inorganic coating to the surfaceof the solid core.

In some further embodiments, the surface treatment comprises applyingone or more layers of a chemical composition different from that of thesolid core.

In some further embodiments, the method comprises derivatizing orfunctionalizing the nanoparticle by attaching one or more chemicalmoieties to the outer surface of the inorganic coating.

According to a further aspect a pharmaceutical formulation is provided,comprising a plurality of nanoparticles according to the invention.

According to a still further aspect, a nanoparticle is provided,comprising a solid, biologically active compound, said nanoparticlehaving an inorganic surface coating, for use in therapy.

According to a still further aspect, a kit is provided, comprising ananoparticle according to the invention.

Further aspects and embodiments of the invention will be apparent fromthe following description and the appended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a nanoparticle of the invention, having a corecomprising a biologically active substance, said core being surroundedby an inorganic coating.

FIG. 2 illustrates another nanoparticle of the invention, having a corecomprising a biologically active substance, said core being surroundedby an inorganic coating, and an intermediate layer between the core andcoating, said intermediate layer having a chemical composition differentfrom that of the core and that of the inorganic coating.

FIG. 3 illustrates another nanoparticle of the invention, having a corecomprising a biologically active substance, said core being surrounded(or enclosed) by an inorganic coating, said coating having chemicalmoieties attached to the outer surface of the inorganic coating.

FIG. 4 illustrates another nanoparticle of the invention, having a corecomprising a biologically active substance, said core being surroundedby an inorganic coating, said coating having chemical moieties attachedto the outer surface of the inorganic coating, the chemical moietiesbeing anchoring groups capable of binding a selected molecule, such as atargeting molecule.

FIG. 5 illustrates a pharmaceutical dosage unit comprising an amount ofone type of nanoparticle according to the invention in combination withan amount of another type of nanoparticle according to the invention.

FIG. 6 illustrates aqueous dissolution profiles of nanoparticlesconsisting of a paracetamol solid core coated by Al₂O₃, prepared by anintermittent agitation method comprising 8 and 5 series, respectively,of applying inorganic material and subsequent agitation; and ofnanoparticles consisting of a paracetamol solid core coated by Al₂O₃,prepared by a method comprising 1 serie of applying inorganic materialand subsequent agitation. The graphs are identified as follows: FilledDiamond 1: 8 series; Square 2: 5 series; Triangle 3: 1 serie.

DETAILED DESCRIPTION OF THE INVENTION

The nanoparticle of the invention is comprised of solid core formed byor comprising a biologically active substance, said core beingsurrounded by an inorganic coating. The inorganic coating may be applieddirectly to the outer surface of the solid core, without anyintermediate layers, or may be applied to one or more intermediatelayers at the surface of the solid core.

The nanoparticle, comprising the core, the inorganic coating andoptionally any intermediate layers in between, has a size, expressed asthe diameter of the particle, generally ranging from a few nanometers,e.g. 1-10 nm, to about 50 μm. In some embodiments, the nanoparticle hassize ranging from 10 nm, or 20 nm, or 40 nm, to 1000 nm, or 500 nm, or200 nm, or 100 nm, or 50 nm. For example, the nanoparticle may have asize ranging from 1 nm to 1000 nm, or from 10 nm to 200 nm, e.g. fromabout 50 nm to 200 nm. In some embodiments, the nanoparticle has a sizeof about 100 nm. In some other embodiments, the nanoparticle has a sizeof from about 10 nm to about 100 nm. In still other embodiments, thenanoparticle has a size of from about 100 nm to about 50 μm, e.g. about1 μm to about 50 μm, or about 10 μm to about 50 μm, such as about 20 μmto 50 μm. The particle size may be determined using methodology andinstrumentation well-known to the person of ordinary skill within thefield, e.g. instruments as sold by Malvern Instruments Ltd.

The form of the nanoparticle may suitably be spherical or essentiallyspherical, but any other form is also possible, e.g. irregular, needleshaped or cuboid shaped, essentially depending e.g. on the method ofpreparation of the nanoparticle core. For a non-spherical particle, thesize may be indicated as the size of a corresponding spherical particleof e.g. the same weight, volume or surface area. An advantageous featureof the method of the invention is the possibility of encapsulating alsoparticles of very irregular shapes, and even particles having pores,crevices etc.

In some embodiments, the coating is applied directly onto the particlecore. In some other embodiments, the coated nanoparticle comprises oneor more, e.g. 1-3, intermediary layers between the coating and the core.For example, the coated nanoparticle may comprise a core, surrounded byan intermediary layer, which in turn carries the inorganic coating,comprising one or more layers of inorganic material(s).

The Solid Core

The nanoparticle of the invention comprises a solid core comprising atleast one biologically active substance (also referred to asbiologically active ingredient), optionally in admixture with one ormore other substances, e.g. excipients or other biologically activeingredients.

In some preferred embodiments, the nanoparticle solid core isessentially comprised of only biologically active substance(s), i.e. itis free from excipients and any other non-biologically activesubstances. In some embodiments, the nanoparticle core is essentiallycomprised of one biologically active substance, e.g. in a crystalline oramorphous state.

The biologically active substance may be selected from any substancewhich preferably is in solid state, or which may be brought to solidstate, at ambient (e.g. room) temperature, e.g. as a crystalline oramorphous material, optionally in combination (e.g. as an admixture orcomplex) with another substance.

As used herein, the term “biologically active substance” or similarexpression, such as “biologically active ingredient” generally refers toany agent, or drug, capable of having a physiologic effect (e.g., atherapeutic or prophylactic effect) on a living subject, e.g. atherapeutically active substance. It also may refer to e.g. a diagnosticagent with no direct therapeutic activity per se, such as a contrastagent for bioimaging.

A biologically active substance according to the invention can beselected e.g. from analgesics, anesthetics, anti-inflammatory agents,anthelmintics, anti-arrhythmic agents, antiasthma agents, antibiotics,anticancer agents, anticoagulants, antidepressants, antidiabetic agents,antiepileptics, antihistamines, antitussives, antihypertensive agents,antimuscarinic agents, antimycobacterial agents, antineoplastic agents,antioxidant agents, antipyretics, immunosuppressants, immunostimulants,antithyroid agents, antiviral agents, anxiolytic sedatives (hypnoticsand neuroleptics), astringents, bacteriostatic agents, beta-adrenoceptorblocking agents, blood products, blood substitutes, bronchodilators,buffering agents, cardiac inotropic agents, chemotherapeutics, contrastmedia, corticosteroids, cough suppressants (expectorants andmucolytics), diagnostic agents, diagnostic imaging agents, diuretics,dopaminergics (antiparkinsonian agents), free radical scavenging agents,growth factors, haemostatics, immunological agents, lipid regulatingagents, muscle relaxants, proteins, peptides and polypeptides,parasympathomimetics, parathyroid calcitonin and biphosphonates,prostaglandins, radio-pharmaceuticals, hormones, sex hormones,anti-allergic agents, appetite stimulants, anoretics, steroids,sympathomimetics, thyroid agents, vaccines, vasodilators, and xanthines.

In some embodiments, the biologically active substance is a poorly watersoluble drug. Non-limiting examples of poorly water soluble drugs whichmay be used according to the present invention are alprazolam,amiodarone, amlodipine, astemizole, atenolol, azathioprine, azelatine,beclomethasone, budesonide, buprenorphine, butalbital, carbamazepine,carbidopa, cefotaxime, cephalexin, cholestyramine, ciprofloxacin,cisapride, cisplatin, clarithromycin, clonazepam, clozapine,cyclosporin, diazepam, diclofenac sodium, digoxin, dipyridamole,divalproex, dobutamine, doxazosin, enalapril, estradiol, etodolac,etoposide, famotidine, felodipine, fentanyl citrate, fexofenadine,finasteride, fluconazole, flunisolide, flurbiprofen, fluvoxamine,furosemide, glipizide, gliburide, ibuprofen, isosorbide dinitrate,isotretinoin, isradipine, itraconazole, ketoconazole, ketoprofen,lamotrigine, lansoprazole, loperamide, loratadine, lorazepam,lovastatin, medroxyprogesterone, mefenamic acid, methylprednisolone,midazolam, mometasone, nabumetone, naproxen, nicergoline, nifedipine,norfloxacin, omeprazole, paclitaxel, phenyloin, piroxicam, quinapril,ramipril, risperidone, sertraline, simvastatin, sulindac, terbinafine,terfenadine, triamcinolone, valproic acid, zolpidem, or pharmaceuticallyacceptable salts of any of these.

The person of ordinary skill in the art will be well acquainted withmethods for preparing the solid core nanoparticles within the desiredsize range. This can be performed using nanoparticle growth in wet ordry conditions or by post synthesis manipulation to achieve a nanopowdere.g. using attrition technology such as use of the pearl/ball milling;or by high-pressure homogenization, spray drying, etc., cf. e.g. theRewiew article: Sivasankar, Mohanty, and B. Pramod Kumar. “Role ofnanoparticles in drug delivery system.” International Journal ofResearch in Pharmaceutical and Biomedical Sciences 1 (2010).

The Inorganic Coating

The nanoparticle comprises an inorganic coating typically in thethickness range of 0.1 nm to 5000 nm, e.g. 0.1 nm to 500 nm, or 0.1 nmto 100 nm. For example, the coating may have a thickness ranging from0.1 to 50 nm, or from 0.2 to 20 nm, e.g. from 0.5 to 10 nm. The coatingmay be of an essentially uniform thickness over at least part of thesurface area of the nanoparticle. In cases where contact holes areformed and covered, the thickness of the coating may vary, e.g. withinthe above indicated limits.

The coating comprises one or more metals, or metal containing compounds,e.g. metal oxide, metal nitride, metal sulphide, metal selenide, metalcarbonates and other ternary compounds etc, and/or one or moremetalloids or metalloid containing compounds. For example, the coatingmay comprise an alkaline metal, an alkaline earth metal, a noble metal,a transition metal, a post-transition metal, or a metalloid, or amixture of any of these, and/or a compound containing any of these.

While in principle all kinds of coatings such as oxide coating, nitridecoating or sulphide coating can be applied to nanoparticle, in a drugdelivery application, the coating material preferably should beessentially non-toxic at the amount of nanoparticles administered, andtherefore, metal or metalloid oxides generally are preferred.

In some embodiments of the invention the nanoparticle comprises at leastone metal oxide layer as an inorganic coating. For example, thenanoparticle may comprise one or several layers of one or more metaloxides. In one embodiment a nanoparticle is provided having a coatingthat comprises one or more layers, wherein each layer essentiallyconsists of one metal oxide.

In some embodiments, the nanoparticle coating comprises one or morelayers composed of mixtures of two or more metal oxides or metalloidoxides. Mixtures of different metal or metalloid oxides in one layer canbe used to modify the properties of the layer and to adapt it to thespecific demands. Accordingly, another preferred embodiment of theinvention is directed to a coated solid nanoparticle comprising abiologically active substance, wherein the coating comprises one or morelayers, wherein each layer essentially consists of a mixture of two ormore metal or metalloid oxides.

In principle, if the coating comprises more than one layer, each of suchlayers can be composed of a different metal or metalloid oxide and/or adifferent mixture of two or more metal or metalloid oxides

Advantageously, the metal or metalloids being present in the coatingis/are aluminium, titanium, magnesium, iron, gallium, zinc, zirconiumand/or silicon, e.g. aluminium, titanium and/or zinc.

Accordingly, in one embodiment, the nanoparticle of the invention iscoated with one or more layers containing metals or metalloids, e.g. inthe form of oxides or hydroxyoxides, selected from aluminium, titanium,magnesium, iron, gallium, zinc, zirconium, niobium, hafnium, tantalum,lanthanum, and/or silicon; e.g. from aluminium, titanium, magnesium,iron, gallium, zinc, zirconium, and/or silicon, e.g. aluminium, titaniumand/or zinc.

More specifically, the present invention is further directed to a coatedsolid nanoparticle, wherein the metal or metalloid oxide/s is/areselected from the group consisting of aluminium oxide (Al₂O₃), titaniumdioxide (TiO₂), iron oxide (Fe_(x)O_(y), e.g. FeO and/or Fe₂O₃ and/orFe₃O₄) or a precursor of iron oxide, such as ferrocene (Fe(C₅H₅)₂), ironcarbonyl (Fe(CO)₅),tris(2,2,6,6-tetramethyl-3,5-heptanedionato)iron(III),(dimethylaminomethyl)ferrocene; gallium oxide (Ga₂O₃), magnesium oxide(MgO), zinc oxide (ZnO), niobium oxide (Nb₂O₅), hafnium oxide (HfO₂),tantalum oxide (Ta₂O₅), lanthanum oxide (La₂O₃), zirconium dioxide(ZrO₂) and/or silicon dioxide (SiO₂).

In some embodiments, the metal or metalloid oxide/s is/are selected fromthe group consisting of aluminium oxide (Al₂O₃), titanium dioxide(TiO₂), iron oxide (FexOy), gallium oxide (Ga₂O₃), magnesium oxide(MgO), zinc oxide (ZnO), zirconium dioxide (ZrO₂) and/or silicon dioxide(SiO₂), e.g. from the group consisting of aluminium oxide (Al₂O₃),titanium dioxide (TiO₂) and zinc oxide (ZnO).

In some embodiments, the coating consists essentially of Al₂O₃. In someother embodiments, the coating consists essentially of TiO₂.

The coating preferably is applied to the nanoparticle core, optionallywith one or more intermediary layers at the surface by a gas phasetechnique, which may be either a chemical or physical technique, such asphysical vapour deposition (PVD), atomic layer deposition (ALD) alsoreferred to as atomic layer epitaxy (ALE), or other similar techniques,e.g. chemical vapour deposition (CVD).

In a preferred embodiment, ALD is used to coat the nanoparticle core,optionally with one or more intermediary layers at the interface. ALD isa gas phase technique wherein an inorganic coating can be formed on allkinds of surfaces and geometries. One great advantage of ALD is itspossibility to completely cover objects with the coating that isapplied. In international patent application WO2012/116814, the contentsof which is incorporated herein by reference, the use of ALD isdisclosed for the coating of solid pharmaceutical preparations, such aspellets, granules, tablets and capsules.

Using ALD, it is possible to obtain ultrathin coatings by deposition ofthe coating material as monomolecular layers. Depending on the number ofreaction cycles one or more atomic layers may be deposited, and coatingsof about 0.01 nm to up to about 0.3 nm in thickness per reaction cyclemay be deposited.

The ALD coating is formed in a series of two or more self-limitedreactions and layers of the coating material may be applied in sequenceuntil a desired coating thickness is achieved.

The coating may be applied at process temperatures from about 20° C. toabout 800° C., or from about 40° C. to about 200° C., e.g. from about40° C. to about 150°, such as from about 50° C. to 100° C. The optimalprocess temperature depends on the reactivity and/or melting point ofthe core substance.

In most instances, the first of the consecutive reactions will involvesome functional group or free electron pairs or radicals at the surfaceto be coated, such as a hydroxy group (—OH) or a primary or secondaryamino group (—NH₂ or —NHR where R e.g. is an aliphatic group, such as analkyl group). The individual reactions are advantageously carried outseparately and under conditions such that all excess reagents andreaction products are removed before conducting the subsequent reaction.

In some embodiments, the biologically active substance may be admixedwith a suitable excipient providing the required functionality, e.g. astarch or cellulosic derivative.

Also, the surface of the nanoparticle may be chemically activated priorto applying the inorganic coating, e.g. by treating the nanoparticlewith hydrogen peroxide, ozone or by applying a plasma treatment, inorder to create free oxygen radicals at the surface of the particle.Thus, in some embodiments, the uncoated nanoparticles, or particlescoated with one or more intermediary layers, are immersed in a hydrogenperoxide containing solution prior to applying the inorganic coating.

Before initiating the reaction sequence, the nanoparticle may be treatedto remove volatile materials that may be absorbed onto its surface, e.g.by exposing the nanoparticle to vacuum and/or elevated temperature.

Oxide coatings can be prepared on nanoparticles having surface hydroxylgroups (—OH) and/or amine groups (—NH₂ or —NHR, where R e.g. is analiphatic group, such as an alkyl group) using the following binary (AB)reaction sequence:

Z—Y—H*+M₁X_(n)→Z—Y-M₁X*+HX  (A1)

Z—Y-M₁X*+H₂0→Z—Y-M₁OH*+HX  (B1)

The above reactions are not balanced, and are only intended toillustrate the reactions at the surface of the particle (i.e. not inter-or intralayer reactions). In the reaction scheme, the asterisk (*)indicates the atom that resides at the surface of the particle orcoating and Y represents oxygen or nitrogen (NH or NR). In the reactionsequence, M₁X_(n) is the precursor, wherein M₁ is the metal or metalloidatom and X is a displaceable nucleophilic group, e.g. a halogen, such asCl or Br, or an alkoxy group, such as methoxy.

Specific compounds having the structure M₁X_(n) that are of particularinterest are silicon tetrachloride (SiCl₄), tetramethylorthosilicate(Si(OCH₃)₄), tetraethyl-orthosilicate (Si(OC₂H₅)₄), trimethyl aluminium(Al(CH₃)₃), triethyl aluminium (Al(C₂H₅)₃), other trialkyl aluminiumcompounds, bis(ethylcyclopentadienyl) magnesium (Mg(C₅H₄ C₂H₅)₂),titanium tetraisopropoxide (Ti{OCH(CH₃)₂}₄) and the like.

Specifically preferred are such precursors which allow for conductingthe atomic layer deposition at low temperatures, e.g. under 100° C. Suchpreferred precursors include trimethyl aluminium (Al(CH₃)₃),bis(ethylcyclopentadienyl) magnesium (Mg(C₂H₅C₅H₄)₂) and titaniumtetraisopropoxide (Ti{OCH(CH₃)₂}₄), titanium tetrachloride (TiCl₄) ordiethyl zinc (Zn(C₂H₅)₂). Therefore, according to one embodiment of theinvention the precursor/s is/are a titanium precursor such as trimethylaluminium, a magnesium precursor such as bis(ethylcyclopentadienyl)magnesium and/or a titanium precursor such as titanium tetraisopropoxideand titanium tetrachloride or diethyl zinc.

In reaction A1, the precursor M₁X_(n) reacts with one or more Z—Y—H*groups on the surface of the nanoparticle to create a new surface grouphaving the form -M₁-X*. M₁ is bonded to the nanoparticle through one ormore Y atoms. The -M₁-X* group represents a site that can react withwater in reaction B1 to regenerate one or more hydroxyl groups. Thegroups formed in reaction B1 can serve as functional groups throughwhich reactions A1 and B1 can be repeated, each time adding a new layerof M₁ atoms. In some cases (such as, e.g., when M₁ is silicon,zirconium, titanium, zincum or aluminium) hydroxyl groups can beeliminated as water, forming M₁-O-M₁ bonds within or between layers.This condensation reaction can be promoted if desired by, for example,annealing at elevated temperatures and/or reduced pressures, cf. alsothe description in international patent application WO2012/116814 andreferences cited therein, i.e. A. C. Dillon et al, Surface Science 322,230 (1995); A. W. Ott et al., Thin Solid Films 292, 135 (1997);Tsapatsis et al. (1991) Ind. Eng. Chem. Res. 30:2152-2159 and Lin etal., (1992), AIChE Journal 38:445-454, which references are allincorporated herein by reference. Following teachings in the abovereferences, coatings of SiO₂, Al₂O₃, ZrO₂, TiO₂ and B₂O₃ may beprepared.

In the foregoing reaction sequences, suitable metals and metalloidsinclude silicon, aluminium, titanium, zinc, magnesium and zirconium.Suitable replaceable nucleophilic groups will vary somewhat with M1, butinclude, for example, fluoride, chloride, bromide, alkoxy, alkyl,acetylacetonate, and the like.

Following ALD as described performance of one cycle results indeposition of one monomolecular layer on the pharmaceutical preparation.If subsequent cycles or series of cycles are performed and the sameprecursor or different precursors, which contain the same metal, areused in each of these cycles or series of cycles, the whole coating iscomposed of the same material, which preferably is a metal or metalloidoxide.

The invention is also directed to a method for producing the coatednanoparticle as described herein, the method comprising applying one ormore layers of inorganic material to a plurality of said solid cores,and submitting said solid cores to intermittent or continuousdisaggregation treatment during or in between application of inorganicmaterial.

It should be noted that according to the present invention, theapplication of inorganic material to the nanoparticles is a gas phasedeposition method. Thus, the particles are not present in a liquid phaseor medium during the application of inorganic material.

In one embodiment, the method comprises (a) introducing into a reactorcontaining the solid nanoparticles to be coated, which is in a gaseousstate, (b) purging and/or evacuating the reactor to remove thenon-reacted precursors and the gaseous reaction by-products (c) exposingof the second precursor to activate the surface again for the reactionof the first precursor (d) purging and/or evacuating of the reactor andoptionally repeating the steps (a) to (d) in order to achieve thedesired coating thickness.

As noted herein above, some action must also be taken either to avoid orto compensate for the holes (disruptions) that may result from contactbetween individual particles during the coating process. Basically, thecontact holes may be avoided by keeping the particles in motion duringat least part of the coating process, e.g. by use of a fluidized bed, orby submitting the particles to an agitation treatment between sequentialapplications of inorganic material to the particles, so as to obtain adisaggregation of any particle aggregates formed during application ofcoating, and/or so as to obtain a rearrangement of particles.

By agitation, as referred to herein, is meant the action of impartingsome spatial reorganization of the nanoparticles relative to each other,either continuously as in a fluidized bed, where the particles may beheld in more or less constant movement, or intermittently, as in the useof one or more steps of sonication in between consecutive steps ofapplication of inorganic material. The idea is to achieve a spatialrearrangement of the particles with respect to each other, and theskilled person will be able to devise various alternative ways ofachieving this without departing from the scope of the presentinvention.

For example, a convenient method for applying the inorganic coating tothe solid is to form a fluidized bed of the nanoparticles, and then passthe various reagents in turn through the fluidized bed under reactionconditions. Methods of fluidizing solid particulate material are wellknown, and generally include supporting the solid material on a porousplate or screen. A fluidizing gas is passed upwardly through the plateor screen, lifting the material somewhat and expanding the volume of thebed. With appropriate expansion, the solid material behaves much as afluid. Fluid (gaseous or liquid) reagents can be introduced into the bedfor reaction with the surface of the solid nanoparticles. The fluidizinggas also may act as an inert purge gas for removing unreacted reagentsand volatile or gaseous reaction products. In this method, contact holesare avoided by the movement of the nanoparticles.

In addition, the reactions can be conducted in a rotating cylindricalvessel or a rotating tube or a reactor chamber with parts that vibrateto keep the particles in movement.

When the particles are coated in a fluidized bed, in a rotatingcylindrical vessel or in a rotating tube, or in other apparatus allowingfor a more or less random movement of the particles during applicationof the material, the occurrence of contact holes may be essentiallyavoided. However, as will be shown in the Examples, and as discussedherein, the uneven coating thickness of particles coated in a methodcomprising intermediary agitation in between two or more coating steps,in itself may lead to a very advantageous release profile.

In a method where essentially no contact holes are formed, i.e. a methodwhere particles are kept in movement relative to each other duringapplication of the inorganic coating, the coating will have a more eventhickness. In such case, the release profile of any one nanoparticlecomposition or formulation may be suitably adapted by combiningnanoparticles of different coating thicknesses and/or of differentcoating materials.

The Intermediary Layer

Prior to applying the inorganic coating, the nanoparticle core may besubjected to one or more preparatory surface treatments. Thus, one ormore intermediary layers of various chemical components may be appliedto the surface of the nanoparticle core, e.g. to protect the core fromunwanted reactions with precursors during the vapour depositiontreatment, to enhance the coating efficiency, or to reduce agglomerationof the nanoparticle cores. It should be realized that while theinorganic coating should provide the nanoparticle with an essentiallycomplete cover, the intermediate layer need not do so.

For example, an intermediary layer may comprise one or more surfactants,in order to reduce agglomeration of the particles to be coated andprovide a hydrophilic surface suitable for the subsequent coating.Surfactants that may be applied for this kind of use are well-known tothe person of ordinary skill in the art, and may be as well non-ionic,as anionic, cationic or zwitterionic. In some embodiments, thesurfactant is a non-ionic surfactant, such as those found in the Tweenseries, e.g. Tween 80.

Thus, in some embodiments, the method of producing a nanoparticleaccording to the invention comprises a step of surface treatment of thenanoparticle core, prior to the application of the inorganic coating,and this surface treatment may comprise the application of a surfactantto the surface of the core. Such application of a surfactant may beachieved by admixing a surfactant with a liquid phase containing thenon-coated nanoparticles (i.e. the solid cores), followed by alyophilisation, spray drying or other drying method, to providenanoparticle cores with a surfactant surface layer.

Another example when a preparatory surface treatment of the nanoparticlecore may be suitable is when an active ingredient, present in thenanoparticle core, is susceptible to reaction with precursor compoundspresent in the gas phase during the gas phase coating process (e.g. theALD process). In such cases, an intermediate layer, e.g. a surfactantlayer, also may serve the purpose of protecting the substances from suchreaction.

Derivatization or Functionalization of the Nanoparticle

In the nanoparticle of the invention, the core containing activeingredient(s) suitably is entirely covered by the inorganic coating. Itis contemplated that the outer surface of the inorganic coating, may bederivatized or functionalized, e.g. by attachment of one or morechemical moieties to the outer surface of the coating, e.g. a compoundor moiety of a compound that enhances the targeted delivery of thenanoparticles in the body of the subject (e.g. a mammal, such as ahuman) to which the nanoparticles are administered. Such compound e.g.may be a polymer, peptide, an antibody, etc.

In some embodiments, the chemical moiety is an anchoring group or“handle”, such as a group containing a silane function. Silanization ofa metal and e.g. metal oxide of hydroxyoxide surface is a well-knownmethod for attaching functional groups to such a surface, and examplesare described e.g. by Herrera A. P. et al., in J. Mater. Chem., 2008,18, 3650-3654, and in U.S. Pat. No. 8,097,742 B2.

To this anchoring group, any desired compound, e.g. the desiredtargeting compound may be attached. Thus, in some embodiments, ananoparticle according to the invention is provided, having a solidcore, optionally one or more intermediary layers, said core and optionalintermediary layers covered by an inorganic coating, wherein anchoringgroups are attached to the outer surface of said inorganic coating, saidanchoring groups being capable of binding at least one targetingmolecule. The binding of the targeting molecule may be accomplished bycovalent binding or by non-covalent binding, e.g. ionic binding,hydrogen bonding or van der Waals bonding, or a combination of differenttypes of binding.

The nanoparticles with anchoring groups will provide a versatile toolfor targeted delivery to various parts of the body, by attaching anappropriate targeting molecule to said nanoparticle. As noted already,such targeting molecule e.g. may be a polymer, peptide, a protein, anucleic acid. For example, the targeting molecule may be an antibody orantibody fragment, or a receptor-binding protein or peptide.

In one further embodiment, a kit is provided, comprising nanoparticlesas described herein, having anchoring groups, e.g. groups containingsilane functions, attached to the outer surface of the inorganiccoating. In some embodiments, such kit also contains at least onereagent useful for attaching target molecules to said anchoring groupsand optionally an instructional material, e.g. a leaflet with writteninstructions of how to use the kit. In some embodiments, the kit alsocontains at least one targeting molecule suitable for being attached tothe nanoparticle via the anchoring group.

The Pharmaceutical Formulation and its Use

According to one aspect, the present invention relates to a nanoparticleas defined herein for use in the medical field, e.g. in therapy or as adiagnostic tool. By means of the coated nanoparticle of the invention,it will be possible to formulate a large diversity of pharmaceuticallyactive compounds, including poorly soluble compounds that previouslyhave been difficult to formulate correctly or that may have beenhampered by poor biodistribution. For example, depending on the activeingredient included in the nanoparticle of the invention, thenanoparticles of the invention may be used in the treatment disorders,such as various types of cancers, inflammatory disorders,neurodegenerative disorders, autoimmune disorders etc.

Thus, according to one aspect, the invention relates to a pharmaceuticalformulation comprising a plurality of nanoparticles according to theinvention. The formulation may be suitable for topical or systemic,parenteral or enteral, e.g. oral or rectal, administration and comprisesa therapeutically effective amount of nanoparticles according to theinvention, where each nanoparticle consists of an active ingredientforming or included in a nanoparticle core surrounded by an inorganiccoating as defined herein. The pharmaceutical formulation in additionmay comprise a pharmaceutically acceptable excipient, e.g. apharmaceutically acceptable carrier for the nanoparticles. In generalthe therapeutically effective amount will vary depending on the activeingredient included in the nanoparticles, the disease state beingtreated, the severity of the disease treated, the age and relativehealth of the subject, the route and form of administration, thejudgment of the attending medical or veterinary practitioner, etc.

As used herein, an “effective amount” of nanoparticles or ofbiologically active agent is that amount effective to bring about thephysiological change desired in the subject to which the nanoparticlesare administered. The term “therapeutically effective amount” as usedherein, means that amount of nanoparticles or of biologically activeagent, alone or in combination with another agent according to theparticular aspect of the invention, that elicits the biological ormedicinal response in the subject to which the nanoparticles of theinvention are administered, e.g. alleviation of the symptoms of thedisease or disorder being treated, or curing or preventing the diseaseor the disorder.

The pharmaceutical formulation of the invention may comprisenanoparticles of different type. For example, a pharmaceuticalformulation may comprise particles of different sizes, e.g. an amount ofnanoparticles having a size within one size range in combination with anamount of nanoparticles having a size within another size range. Thedifferent size ranges may be due to different sizes of the cores, ordifferent thicknesses of the coatings, or a combination of both.

Thus, in some embodiments, there is provided a pharmaceuticalformulation comprising an amount of nanoparticles having a thininorganic coating, or even having no inorganic coating, in combinationwith an amount of nanoparticles having a thicker inorganic coating. Bycombining, in one and the same formulation, nanoparticles with differentcoating thicknesses and/or different core sizes, the drug releaseobtained in the body of the treated subject may be extended over aselected time period, e.g. from nearly instant release to prolongedrelease.

In some embodiments, therefore, the pharmaceutical formulation comprisesnanoparticles of different active ingredients and/or of differentrelease profiles. For example, in some embodiments, the formulationcomprises an amount of nanoparticles having a coating of a thicknessthat allows for release of a first substance over a first period oftime, and an amount of nanoparticles having a coating of another,greater, thickness that allows for release of a second substance over asecond period of time, which second period may be overlapping with thefirst period of time or not.

In other embodiments, the pharmaceutical formulation of the inventioncomprises nanoparticles having different functionalization orderivatization at the nanoparticle surface. For example, apharmaceutical formulation of the invention may comprise an amount ofnanoparticles functionalized with one type of targeting molecule,directing the nanoparticles to one organ or cell type in the body, andan amount of nanoparticles functionalized with another type of targetingmolecule, directing the nanoparticles to the same or another organ orcell type in the body.

For enteral, e.g. oral, administration, the nanoparticles of theinvention may be formulated in a wide variety of dosage forms. Thepharmaceutically acceptable carriers may be either solid or liquid.Solid form preparations include granules (wherein each granule consistsof several nanoparticles and e.g. a binder), tablets, pills, lozenges,capsules, cachets, suppositories. A solid carrier may be one or moresubstances which may also act as e.g. diluents, flavouring agents,lubricants, binders, preservatives, tablet disintegrating agents, or anencapsulating material. In tablets, the nanoparticles of the inventiongenerally are mixed with the carrier having the necessary bindingcapacity in suitable proportions and compacted in the shape and sizedesired. Suitable carriers include but are not limited to magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatine, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.

In another embodiment, the nanoparticles are encapsulated e.g. in a softor hard shell capsule, e.g. a gelatine capsule.

Exemplary compositions for rectal administration include suppositorieswhich can contain, for example, a suitable non-irritating excipient,such as cocoa butter, synthetic glyceride esters or polyethyleneglycols, which are solid at ordinary temperatures, but liquefy and/ordissolve in the rectal cavity to release the nanoparticles.

The nanoparticles of the invention also may be administeredparenterally, e.g. by inhalation, injection or infusion, e.g. byintravenous, intraarterial, intraosseous, intramuscular, intracerebral,intracerebroventricular, intrasynovial, intrasternal, intrathecal,intralesional, intracranial, intratumoral, intracutaneous andsubcutaneous injection or infusion.

Thus, for parenteral administration, the pharmaceutical compositions ofthe invention may be in the form of a sterile injectable or infusiblepreparation, for example, as a sterile aqueous or oleaginous suspensionof the inventive nanoparticles. This suspension may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents (e.g., Tween 80), and suspending agents. The sterileinjectable or infusible preparation may also be a sterile injectable orinfusible suspension in a non-toxic parenterally-acceptable diluent. Forexample, the pharmaceutical composition may be a solution in1,3-butanediol. Other examples of acceptable vehicles that may beemployed in the compositions of the present invention include, but arenot limited to, mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils may be employed as asuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or diglycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil suspensions may also contain a long-chain alcohol diluent ordispersant.

In some embodiments, the pharmaceutical composition of the invention isa formulation suitable for inhalation, e.g. an inhalation powder such asmay be administered by use of dry powder inhalers. This type offormulations are described e.g. by Kumaresan C., et al in PharmaTimes—Vol. 44—No. 10—October 2012, pp 14-18 and in by Mack P., et al.,in Inhalation, vol. 6, No. 4, August 2012, pp. 16-20, the contents ofwhich are incorporated herein by reference.

In such embodiments, the size of the nanoparticle suitably is in therange having a diameter of from about 20 to 50 μm.

In some embodiments, of an inhalation formulation of the invention, thedrug release from the formulation may be controlled so as to give in oneand the same dosage, immediate or quick release and prolonged release.This is achieved by combining particles having different coatings, e.g.varying e.g. in thickness, and optionally also particles withoutcoating. For example, particles having no coating may be combined withparticles having a coating of e.g. 5 nm, which after inhalation start todissolve after e.g. 30 minutes, and optionally also particles with aneven thicker coating, which after inhalation start to dissolve after amuch longer time period, e.g. of several days.

The pharmaceutical compositions of the invention also may beadministered topically, to the skin or to a mucous membrane. For topicalapplication, the pharmaceutical composition may be e.g. a lotion, a gel,a paste, a tincture, a transdermal patch, a gel for transmucosaldelivery, containing the nanoparticles of the invention. The compositionmay be formulated with a suitable ointment containing the inventivenanoparticles suspended in a carrier, such as mineral oil, liquidpetroleum, white petroleum, propylene glycol, polyoxyethylenepolyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical composition may be formulated with a suitable lotion orcream containing the nanoparticles of the invention in a carrier.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetaryl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation.

Suitable pharmaceutical excipients, e.g. carriers, and methods ofpreparing pharmaceutical dosage forms are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in art of drug formulation.

In some embodiments, a pharmaceutical composition of the invention maycomprise from approximately 1% to approximately 99%, preferably fromapproximately 20% to approximately 90% of nanoparticles of theinvention, together with at least one pharmaceutically acceptableexcipient.

The nanoparticle of the invention and its use are further illustrated inthe accompanying drawings. In these drawings, FIG. 1 represents ananoparticle 1 comprising a solid core 2 enclosed by a coating 3. Whilethe particle represented in FIG. 1 is spherical, it should be realizedthat this is of course merely a schematic representation, and in factthe nanoparticle 1 may also be irregular in shape. Likewise, theproportions between the nanoparticle diameter and the coating thicknessare merely illustrative and these proportions may vary depending onfactors such as the desired dissolution time, the numbers of layers inthe coating, etc.

The nanoparticle 1 represented in FIG. 2 comprises an intermediary layer4, between the core 2 and the coating 3. This intermediary layer 4 mayenclose and cover entirely the core 2 or not, depending e.g. on thepurpose of the layer, which is illustrated by the broken line in FIG. 2.

The nanoparticle 1 represented in FIG. 3 comprises chemical moieties 5attached to the outer surface of the inorganic coating 3, e.g. anchoringgroups permitting to attach another molecule to the surface, such as atargeting molecule. The chemical moiety 5 also may be a moietyfulfilling in itself a purpose such as targeting of an organ or celltype in the body of a subject to which the nanoparticle is administered.

While the nanoparticle 1 shown in FIG. 3 does not comprise anyintermediary layer of the type illustrated in FIG. 2, it should berealized that such variant is also contemplated.

In FIG. 4, a nanoparticle 1 is shown having a chemical moiety 5 which isan anchoring group, to which anchoring group a selected molecule 6 isbound. As for the nanoparticle of FIG. 3, it is contemplated a variantof the nanoparticle of FIG. 4 comprises at least one intermediary layerbetween the inorganic coating 3 and the solid core 2.

In the embodiments represented in FIGS. 1-4, the inorganic coating alsomay comprise several layers of different compositions, e.g. one layer ofAl₂O₃ and one layer of TiO₂.

Finally, in FIG. 5 a pharmaceutical dosage unit 7 according to theinvention is illustrated, containing an amount of a first nanoparticle 1a and an amount of a second nanoparticle 1 b. In FIG. 5, thenanoparticles are schematically shown as differing in size of both coreand coating. It should be realized that also the chemical composition ofthe nanoparticles may differ, as may the surface functionalization. Thedosage unit may be e.g. a capsule. In addition to the nanoparticles ofthe invention the dosage unit may comprise also other activeingredients, in particulate or non-particulate form as well aspharmaceutically acceptable excipients.

The invention will now be further described by the followingnon-limiting examples.

EXAMPLES Example 1

A powder of a drug has grain sizes of 100 nm. A problem with the drug isthat it cannot be administered into a patient orally and maintain itsoptimal medical effect. It would be more favourable if the drug couldreach its destination in the body as a relatively inert nanoparticlebefore the release of the drug. To achieve this, a thin coating ofalumina (Al₂O₃) is applied to the grains/powder using ALD. The aluminaprevents the powder from being dissolved directly in the body and thethickness of the coating determines how long the dissolution will take.Dissolution of alumina is possible by presence of chloride ions even inacidic and neutral pH.

The process to form the alumina coating by ALD is performed at atemperature of 50-100° C. and the drug withstand that temperaturewithout being deteriorated.

Example 2

A powder of a drug has grain sizes of 1000 nm. A problem with the drugis that its surface is terminated by polar functional groups, preventingtransport through cell membranes. By applying a thin coating of titaniumdioxide (TiO₂) on the surface of the grains this problem can becircumvented since titanium oxide provides another chemical compositionof its surface than the drug.

The process to form the titanium oxide coating by ALD is performed at atemperature of 100-150° C. and the drug withstands that temperaturewithout being deteriorated.

Example 3

A powder of a drug has a size of 100 nm. A problem with the drug is thatit cannot be modified with the desired molecules for targeting or otherfunctions. Coating the grains with a thin film of alumina (Al₂O₃) onwhich the desired molecules for targeting or other functions can becoupled circumvents the problem.

Example 4

A powder of a drug has a size of 200 nm. The powder is admixed with anaqueous solution of Tween 80 and allowed to dry. To the powder withsurfactant treated surface, a thin coating of alumina (Al₂O₃) isapplied.

Example 5

A particle powder (0.3 g, approximate particle size 150 nm) offelodipine (i.e. (RS)-3-ethyl 5-methyl4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate)was placed in a 5 cm² tray and the tray was introduced into an ALDreaction chamber. The reactor was heated to a temperature of 50-100° C.ALD was performed using trimethyl aluminium and water as ALD precursors.50 ALD cycles were performed with ALD pulse lengths of 1-10 seconds. Theobtained product consisted of particles having a coating of about 3 nmthickness, except at points of contact between individual particles.

The felodipine particle powder was discharged from the reactor, placedin 5 ml of water and sonicated for 5 minutes. Felodipine has very lowwater solubility, and therefore water was selected as a liquid phase(dispersion medium) for the sonication step. The sonication resulted ina deagglomeration with breaking up of the contact points betweenindividual felodipine particles.

The felodipine particle powder was allowed to settle in the water andexcess water was decanted. The powder was then allowed to dry. Thepowder was “fluffed” using a spatula and placed once again in the trayfor loading into the reactor.

The steps of ALD coating of the powder, sonication of the powder, anddrying of the sonicated powder were repeated 3 times, to a total of 4cycles. The obtained dry powder was free-flowing and the main fraction(more than 50% of the particles) had a 10-20 nm thick aluminum oxidecoating without disruptions (contact holes).

Example 6

A particle powder (0.3 g, approximate particle size 30 μm) ofparacetamol (i.e. N-(4-hydroxifenyl)acetamid) was placed in a 5 cm² trayand the tray was introduced into an ALD reaction chamber. The reactorwas heated to a temperature of 50-100° C. ALD was performed usingtrimethyl aluminium and water as ALD precursors. 50 ALD cycles wereperformed with ALD pulse lengths of 1-10 seconds. The obtained productconsisted of particles having a coating of about 3 nm thickness, exceptat points of contact between individual particles.

The paracetamol particle powder was discharged from the reactor, placedin 5 ml of heptanol and sonicated for 5 minutes. Paracetamol has highwater solubility but low solubility in e.g. heptane, and thereforeheptane was selected as a liquid phase (dispersion medium) for thesonication step. The sonication resulted in a deagglomeration withbreaking up of the contact points between individual paracetamolparticles.

The paracetamol particle powder was allowed to settle and excess heptanewas decanted. The powder was then allowed to dry. The powder was“fluffed” using a spatula and placed once again in the tray for loadinginto the reactor.

The steps of ALD coating of the powder, sonication of the powder anddrying of the sonicated powder were repeated 4 times, to a total numberof 5 cycles. After the last drying step, the obtained powder wasfree-flowing and the main fraction (more than 50% of the particles) hada 10-20 nm thick aluminum oxide coating without disruptions (contactholes).

Example 7

A particle powder (0.03 g, approximate particle size 150 nm) offelodipine is placed in a 5 cm² tray and the tray is introduced into anALD reaction chamber. The reactor is heated to a temperature of 50-100°C. ALD is performed using trimethyl aluminium and water as ALDprecursors. 10-30 ALD cycles are performed with ALD pulse lengths of0.1-450 seconds. The obtained product consists of particles having acoating of about 0.5-2 nm thickness, except at points of contact betweenindividual particles.

The felodipine particle powder is discharged from the reactor, placed in5 ml water and sonicated for 5 minutes.

After 5 minutes of sonication, the powder is allowed to settle andexcess water is decanted. The powder is then allowed to dry. The powderis “fluffed” using a spatula and placed once again in the tray forloading into the reactor.

The steps of ALD coating of the powder, sonication of the powder, anddrying of the sonicated powder are repeated 3 times, i.e. a total of 4series, each series comprising a deposition step including 50 ALDcycles, followed by a step of sonication and drying of the sonicatedparticles. The obtained dry powder is free-flowing and has a 2-8 nmthick aluminum oxide coating without disruptions (contact holes).

The felodipine containing nanoparticles having a non-disrupted coatingcan if necessary be separated from any remaining incompletely coverednanoparticles by soaking of the powder in dichloromethane, which is asolvent for felodipine, separating the soaked particles from the soakingmedium, and optionally rinsing the separated particles.

The precursors are thereafter changed to titanium tetrachloride andwater ALD is performed on the aluminium oxide coated felodipineparticles, using essentially same conditions as in the previous ALDtreatment (reactor temperature 50-100° C., 10-30 ALD cycles, ALD pulselengths of 1-10 seconds), so as to form a coating of titanium oxide ontop of the aluminum oxide coating. In this second sequence of ALDtreatment and deagglomeration treatment, the series are performed intotal 4 times.

Example 8

The procedure of Example 5 is followed, but the series of ALD treatmentand deagglomeration treatment are performed 8 times. A felodipinenanoparticle powder is obtained with a 15 nm thick coating of aluminiumoxide, and a particle size of 40 μm.

Example 9

The procedure of Example 6 was followed, but the series of ALD treatmentand deagglomeration treatment were performed 8 times in total. Aparacetamol nanoparticle powder was obtained, having 15 nm thick coatingof aluminium oxide and a particle size of 40 μm.

Example 10

The procedure of Example 5 is followed, but titanium tetrachloride andwater are used as precursors in the ALD treatment. A felodipinenanoparticle powder is obtained with a 15 nm thick coating of titaniumoxide, and a particle size of 40 μm.

Example 11

The procedure of Example 6 is followed, but titanium tetrachloride andwater are used as precursors in the ALD treatment. A paracetamolnanoparticle powder is obtained with a 5 nm thick coating of titaniumoxide, and a particle size of 150 nm.

Comparative Example 12

The procedure of Example 6 was followed, but after the first sonicationand drying of the ALD treated nanoparticles, the dry powder wascollected, without any further application of inorganic coating.

Example 13

About 15 mg of the coated paracetamol nanoparticles prepared in Example6 were admixed with 100 ml of deionized water and then sonicated for 5minutes. The mixture was diluted to 1 liter by addition of moredeionized water and was stirred using a magnetic stirrer. Samples of thesupernatant (water containing dissolved paracetamol) were taken atregular intervals and the absorbance at 243 nm was measured as anindication of the paracetamol concentration in the supernatant.

Example 14

The procedure of Example 13 was repeated using the coated paracetamolnanoparticles of Example 9.

Comparative Example 15

The procedure of Example 13 was repeated using the paracetamolnanoparticles of Comparative Example 12.

In FIG. 6 the results of the dissolution tests of Examples 13-15 areshown. In FIG. 6, the diamonds 1 show the results obtained in Example 14(8 coating/agitation series); the squares 2 show the results obtained inExample 13 (5 coating/agitation series); and the triangles 3 show theresults obtained in Comparative Example 15 (1 coating/agitation series).The straight, horizontal line with no marking (at about 0.8) representsthe concentration of paracetamol in the aqueous phase at totaldissolution of the coated nanoparticles of Examples 6 and 9.

From the results, it appears that the coated nanoparticles of theinvention have a profile of substantially delayed dissolution. Moreover,the delay may be tailored varying the number of ALD (or otherdeposition) cycles and the number of series comprising ALD (or othercoating) treatment with intermediate agitation treatment.

Without wishing to be bound to any theory, it is considered thatnormally, the release of the solid core from any one particle is aninstantaneous event, occurring after a delay which increases with thethickness of the coating surrounding the solid core (i.e. the thickerthe coating, the longer the time for its disintegration or dissolution).Paracetamol is a very water soluble substance and in theory, therefore,a plurality of nanoparticles having a uniform coating thickness, couldbe expected to show a dissolution profile of paracetamol in an aqueousliquid phase comprising an initial lag time during which the coatingslowly dissolves in the liquid phase, followed by a short period ofrapid increase of the paracetamol concentration in the liquid phase,corresponding to rapid dissolution of paracetamol at the time pointwhere the surrounding coating has dissolved enough to provide access ofthe water to the paracetamol. In contrast to this theoreticaldissolution profile, the nanoparticles of the inventions show a rapidrise, followed by a slow and steady rise, of the paracetamolconcentration in the liquid phase.

The rapid rise of paracetamol concentration during the first few minutesare considered to correspond to remaining incompletely encapsulatednanoparticles, from which paracetamol is quickly dissolved. Thissupposition is supported by the fact the maximum level of the initialrapid increase of paracetamol concentration in the supernatant decreasesas the number of coating series increases. Increasing the number ofseries of coating and deagglomeration will lead to an increased fractionof completely covered nanoparticles.

The subsequent slow rise of paracetamol concentration in the supernatantis considered to be the result of the uneven coating of thenanoparticles. Again without wishing to be bound to any theory, it maybe surmised that during each application of coating, new contact pointsbetween particles will arise in a random manner, and thereby eachparticle will have a coating of varying thickness over its surface.Dissolution of a plurality of such particles having uneven surfacecoatings will result in the slowly rising concentration profile obtainedby Examples 13 and 14. In an in vivo administration, nanoparticles ofExample 6 and 9, will be able to provide a delayed release ofparacetamol in the body of the treated subject.

It should be noted that incompletely coated nanoparticles, such as thosegiving rise to the initial rapid rise in paracetamol in Examples 13 and14, may easily be eliminated from the nanoparticle product obtained atthe end of the coating process, e.g. by bringing the product in contactwith a solvent for the nanoparticle solid core. For example,nanoparticles having a water soluble solid core may be washed with wateror an aqueous solution and allowed to dry. Particles with a non-watersoluble solid core, e.g. a core soluble in organic solvent, may bewashed with such organic solvent. In the washing step(s), the solid coreof the incompletely coated nanoparticles will dissolve and particleshaving a completely enclosed solid core may thereafter be separated fromthe liquid phase, and optionally rinsed and dried.

Thus, in some embodiments the present invention provides a method ofpreparing a plurality of nanoparticles, each particle having a solidcore comprising a biologically active substance, said core beingenclosed by an inorganic coating, the method comprising the steps ofapplying one or more layers of inorganic material to said solid cores,

submitting said solid cores to agitation during and/or in betweenapplication of the layers of inorganic material, andbringing the obtained nanoparticles into contact with a solvent for thesolid core.

In the step of eliminating incompletely coated nanoparticles, thesolvent for the solid core preferably should not be a solvent for theinorganic coating, or should be only a poor solvent for the inorganiccoating.

Very advantageously, by the method of the invention, nanoparticlecompositions may be provided having a desired controlled releaseprofile, e.g. a delayed release profile, a sustained release profileetc. In some embodiments, the method therefore comprises:

bringing a sample of the coated nanoparticles into contact with a liquidphase which is a solvent for the solid core and for the inorganiccoating,measuring dissolution of the coated nanoparticle solid cores in theliquid phase,comparing the dissolution of coated nanoparticle solid cores with thedissolution of similar solid cores having no inorganic coating,determining a delay in dissolution of the coated nanoparticle solidcores compared to the dissolution of similar solid cores having noinorganic coating, andselecting coated nanoparticles having a delay in dissolution exceeding apredetermined length of time.

The liquid phase which is a solvent for the solid core and for theinorganic coating e.g. may be water, an aqueous solution, a phosphatebuffer or any other suitable liquid.

The dissolution of the coated nanoparticles in the liquid phase may bemeasured e.g. by determining the concentration of the active substancein the liquid phase.

The dissolution profile of coated nanoparticle solid cores is comparedto the dissolution profile of similar solid cores having no inorganiccoating in order to provide information about the delay in dissolutionprovided by the coating.

For example, the delay in dissolution may be determined as thedifference between the time needed to dissolve at least 50% by weight,of the coated nanoparticle solid cores and the time needed to dissolveat least 50% by weight of the solid cores having no coating.

In some embodiments, the delay in dissolution is determined as thedifference between the time needed to dissolve at least 90% by weight,of the coated nanoparticle solid cores and the time needed to dissolveat least 90% by weight of the solid cores having no coating.

In some embodiments, the delay in dissolution is determined as thedifference between the time needed to dissolve at least 95% by weight,of the coated nanoparticle solid cores and the time needed to dissolveat least 95% by weight of the solid cores having no coating.

In some embodiments, the delay in dissolution is determined as thedifference between the time needed to dissolve at least 99% by weight,of the coated nanoparticle solid cores and the time needed to dissolveat least 99% by weight of the solid cores having no coating.

In some embodiments, the delay in dissolution is determined as thedifference between the time needed to dissolve 100% by weight, of thecoated nanoparticle solid cores and the time needed to dissolve 100% byweight of the solid cores having no coating.

Coated nanoparticles having a delay in dissolution exceeding apredetermined length of time may thus be suitably selected. If the delayin dissolution is considered insufficient according to the test,parameters of the method, e.g. the number of applications of inorganicmaterial to the nanoparticles may be increased so as to provide athicker coating, or the inorganic material may be changed, etc.

Very advantageously, the present invention thus provides for a method ofobtaining a pharmaceutical formulation with a fine tuning of releaseproperties. Moreover, nanoparticles of different release profiles may becombined in one and the same pharmaceutical formulation, which increaseseven further the possibility of varying the release profile of theformulation.

1.-23. (canceled)
 24. A method of preparing a plurality of coatedparticles of a size that is between 1 nm and 50 μm, said coatedparticles having a solid core comprising a biologically activesubstance, said solid core being enclosed by an inorganic coating; themethod comprising applying more than one layer of inorganic materialcomprising at least one metal or metalloid element to a plurality ofsaid solid cores by an application method selected from atomic layerdeposition, chemical vapour deposition or physical vapour deposition,wherein the inorganic material and/or precursors for forming theinorganic material is/are present in gas phase, and submitting saidsolid cores to intermittent agitation between application of layers ofinorganic material so as to obtain either: (a) disaggregation ofparticle aggregates formed during application of a coating, or (b) aspatial rearrangement of particles.
 25. The method of claim 24,comprising (i) applying inorganic material to a plurality of said solidcores (ii) submitting the plurality of said solid cores to agitation,(iii) repeating step (i) n times, wherein n is an integer of at least 1,and (iv) when n is an integer of at least 2, repeating step (ii) afterat least some of the steps (i).
 26. The method of claim 25, wherein n isan integer of at least 1 and at most
 50. 27. The method of claim 26,wherein n is an integer of at least 2 and at most
 20. 28. The method ofclaim 27, wherein n is an integer of at least 3 and at most
 10. 29. Themethod of claim 24, wherein the element that is a metal or metalloid ispresent as an oxide, a hydroxyoxide, a carbide, a selenide, a nitride,sulphide, fluoride, chloride and/or a salt.
 30. The method of claim 24,wherein the element is present as an oxide.
 31. The method of claim 24,wherein the inorganic material comprises aluminium oxide (Al₂O₃),titanium dioxide (TiO₂), iron oxide (Fe_(x)O_(y)), gallium oxide (Ga₂O₃)and magnesium oxide (MgO), zinc oxide (ZnO), niobium oxide (Nb₂O₅),hafnium oxide (HfO₂), tantalum oxide (Ta₂O₅), lanthanum oxide (La₂O₃),zirconium dioxide (ZrO₂) and/or silicon dioxide (SiO₂).
 32. The methodof claim 24, wherein the inorganic coating comprises aluminium oxide,titanium dioxide and/or zinc oxide.
 33. The method of claim 24, whereinseveral layers of inorganic material are applied to the solid core. 34.The method of claim 24, wherein the biologically active substance is atherapeutically active substance.
 35. The method of claim 24, whereinthe solid core essentially comprises the biologically active agent. 36.The method of claim 24, wherein the inorganic coating surrounds,encloses or encapsulates the biologically active agent sufficientlycompletely to enable a therapeutically effective controlled or delayedrelease of said biologically active agent therefrom.
 37. The method ofclaim 24, wherein the inorganic coating is of an essentially uniformthickness that is in the range between 0.1 and 500 nm.
 38. The method ofclaim 37, wherein the thickness is in the range between 0.1 and 100 nm.39. The method of claim 37, wherein the thickness is in the rangebetween 0.1 and 50 nm.
 40. The method of claim 24, wherein thebiologically active substance is selected from the group: an analgesic,an anesthetic, an anti-inflammatory agent, an anthelmintic, ananti-arrhythmic agent, an antiasthma agent, an antibiotic, an anticanceragent, an anticoagulant, an antidepressant, an antidiabetic agent, anantiepileptic, an antihistamine, an antitussive, an antihypertensiveagent, an antimuscarinic agent, an antimycobacterial agent, anantineoplastic agent, an antioxidant agent, an antipyretic, animmunosuppressant, an immunostimulant, an antithyroid agent, anantiviral agent, an anxiolytic sedative, a hypnotic, a neuroleptic, anastringent, a bacteriostatic agent, a beta-adrenoceptor blocking agent,a blood product, a blood substitute, a bronchodilator, a bufferingagent, a cardiac inotropic agent, a chemotherapeutic, a contrast media,a corticosteroid, a cough suppressant, an expectorant, a mucolytic, adiagnostic agent, a diagnostic imaging agent, a diuretic, adopaminergic, an antiparkinsonian agent, a free radical scavengingagent, a growth factor, a haemostatic, an immunological agent, a lipidregulating agent, a muscle relaxant, a protein, a peptide, apolypeptide, a parasympathomimetic, a parathyroid calcitonin, abiphosphonate, a prostaglandin, a radio-pharmaceutical, a hormone, a sexhormone, an anti-allergic agent, an appetite stimulant, an anoretic, asteroid, a sympathomimetic, a thyroid agent, a vaccine, a vasodilatorand a xanthine.
 41. The method of claim 24, wherein the biologicallyactive substance is selected from the group: alprazolam, amiodarone,amlodipine, astemizole, atenolol, azathioprine, azelatine,beclomethasone, budesonide, buprenorphine, butalbital, carbamazepine,carbidopa, cefotaxime, cephalexin, cholestyramine, ciprofloxacin,cisapride, cisplatin, clarithromycin, clonazepam, clozapine,cyclosporin, diazepam, diclofenac sodium, digoxin, dipyridamole,divalproex, dobutamine, doxazosin, enalapril, estradiol, etodolac,etoposide, famotidine, felodipine, fentanyl citrate, fexofenadine,finasteride, fluconazole, flunisolide, flurbiprofen, fluvoxamine,furosemide, glipizide, gliburide, ibuprofen, isosorbide dinitrate,isotretinoin, isradipine, itraconazole, ketoconazole, ketoprofen,lamotrigine, lansoprazole, loperamide, loratadine, lorazepam,lovastatin, medroxyprogesterone, mefenamic acid, methylprednisolone,midazolam, mometasone, nabumetone, naproxen, nicergoline, nifedipine,norfloxacin, omeprazole, paclitaxel, phenyloin, piroxicam, quinapril,ramipril, risperidone, sertraline, simvastatin, sulindac, terbinafine,terfenadine, triamcinolone, valproic acid, zolpidem and pharmaceuticallyacceptable salts of any of these agents.
 42. The method of claim 24,wherein one or more intermediary layers comprising one or moresurfactants is/are present.
 43. The method of claim 24, wherein theouter surface of the inorganic coating is derivatized or functionalizedby attachment of one or more chemical moieties to the outer surface ofthe coating.
 44. The method of claim 43, wherein the one or morechemical moieties comprises one or more compounds that enhances thetargeted delivery of the nanoparticles in the body of a subject to whomthe formulation are administered.
 45. The method of claim 43, whereinthe one or more chemical moieties comprise an anchoring group or“handle” which contains a silane function to which a targeting moleculemay be attached.
 46. The method of claim 45, wherein the one or morechemical moieties comprises one or more nucleic acids, antibodies,antibody fragments, receptor-binding proteins or receptor-bindingpeptides.
 47. A plurality of coated nanoparticles obtainable by a methodaccording to claim
 24. 48. The plurality of coated nanoparticlesaccording to claim 47, wherein the biologically active substance is atherapeutically active substance.
 49. A pharmaceutical compositioncomprising a plurality of coated nanoparticles according to claim 47 anda pharmaceutically acceptable carrier.
 50. A pharmaceutical compositionaccording to claim 49 in the form of a sterile injectable or infusiblesuspension of particles in a parenterally-acceptable diluent
 51. Apharmaceutical composition according to claim 50, which is in the formof a sterile aqueous or oleaginous suspension.
 52. A method of treatmentof a medical disorder, which method comprises administering to a patientsuffering from or susceptible to said medical disorder a pharmaceuticalformulation according to claim 49, wherein the biologically active agentis effective in the treatment of said medical disorder.